Prostate cancer (PCa) is a heterogeneous malignancy harboring many phenotypically and functionally distinct cancer cells. Androgen receptor (AR) plays an important role in PCa development and progression. Androgen-deprivation therapy (ADT) has been used as the first-line treatment in advanced PCa, but patients generally develop castration resistance within 1-2 years. Castration-resistant PCa (CRPC) is then treated with antiandrogens such as enzalutamide (Enza); however, patients also quickly develop Enza resistance in several months. Both ADT and Enza target AR signaling, whose long-term efficacy depends on PCa cells to express AR. Yet, our work, together with many others', has revealed that untreated PCa harbor not only AR+ but also AR-/lo PCa cells and this AR heterogeneity becomes accentuated in CRPC. Our recent work has directly linked AR heterogeneity to distinct ADT/Enza responses, and further demonstrated that both AR+ and AR-/lo subpopulations of PCa cells may co-evolve under the pressure of ADT/Enza to mediate castration resistance. Critically, most current anti-PCa therapeutic efforts have been directed towards targeting AR+/hi PCa cells, while largely ignoring the AR-/lo cell population. Since AR-/lo cells can also propagate CRPC, simultaneously targeting the AR-/lo population may inhibit and even prevent resistance to Enza and abiraterone acetate. Our pre-clinical modeling using matched androgen-dependent and androgen-independent xenograft models of PCa has identified BCL-2 as a critical driver and also a viable therapeutic target of CRPC. Based on this new knowledge and promising pre-clinical therapeutic studies, we hypothesize that co-targeting AR+/hi (bulk tumor cells) and AR-/lo PCa cells in heterogeneous patient tumors using Enza and the BCL-2 inhibitor venetoclax will achieve superior clinical outcomes. This hypothesis is currently being tested in a new phase Ib/II clinical trial developed by our research group at RPCCC in collaboration with AbbVie (NCT03751436). In support of the objectives for this clinical trial, we propose the following correlative studies in this R21 project, with 3 Aims: Aim 1: To identify patient responders and non-responders to combination Enza/venetoclax therapy; Aim 2: To identify the best treatment related biomarkers associated with patient response to Enza/venetoclax combination therapy; and Aim 3: To establish clinically relevant 3D organoid models to better understand patient responsiveness and tumor heterogeneity. SIGNIFICANCE/IMPACT: We have assembled an outstanding team of basic and physician scientists for the proposed correlative studies. Successful completion of this project will facilitate the development of paradigm- sifting and potentially practice-changing treatment regimens for (Enza-nave) mCRPC patients. Furthermore, the proposed studies will have significant impact on the application of circulating tumor cells to pharmacodynamic modeling of tumor response and their use as a source of novel 3D tumor models. !